Antibodies against a fused ''lacZ-yeast mitochondrial intron'' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.

Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.     

Stimulation of yeast mating hormone activity by synthetic oligopeptides.

The biological activities of two synthetic oligopeptides (His-Trp-Leu-Gln-Leu and Trp-Leu-Gln-Leu), which represent part of the primary structure of the mating hormone alpha factor from Saccharomyces cerevisiae, were studied. The peptides did not exhibit hormonal activity by themselves. However, both intensified the mating-type-specific inhibitory effect of native alpha factor on the division of haploid cells of mating type a. Random peptides or mixtures of the corresponding amino acids did not stimulate alpha factor activity. Likewise, a synthetic peptide representing another part of the alpha factor sequence was ineffective. In addition, the activity of a factor, the mating hormone produced by a cells, was not influenced by the synthetic peptides, indicating that the compounds specifically affect the interaction between alpha factor and its target cells. The analysis of the utilization of the tetrapeptide as a source of amino acids for auxotrophic a strains suggested an extracellular site of action for the observed enhancement of alpha factor activity.     

Role of the 5' hairpin structure in the splicing accuracy of the fourth intron of the yeast cob-box gene

The splicing mechanism of the maturase-coding introns is poorly understood. We have systematically examined the phenotypes of a large number of revertants from the mitochondrial mutation G2457. This mutation results from a single base change near the 5' splicing site. We show here that this base change does not completely block the splicing of the intron but rather affects the specificity of the splicing process. We examine four classes of revertants which allow us to characterize the crucial role of a stem and loop structure in the accuracy of the intron excision process. An unexpected class of revertant suggests that other elements are involved in this mechanism. Reversion of G2457 can also occur via the excision in the mitochondrial genome of the intron coding sequence. These results are discussed in relation to the possible role fulfilled by the maturase in the control of intron splicing.     

Antibodies against synthetic peptides react with the second Epstein-Barr virus-associated nuclear antigen.

Five peptides were synthesized on the basis of amino acid sequences predicted from the transformation-associated BamHI WYH region of the genome of the Epstein-Barr virus (EBV). Antisera to two peptides deduced from a 1.6-kb open reading frame in the BamHI H fragment identified an 87 000-dalton nuclear polypeptide that was present in EBV-carrying cell lines that expressed the second EBV-determined nuclear antigen (EBNA-2). This polypeptide was not detected in cell lines that carried EBV variants with a deleted BamHI WYH region or in EBV-negative cell lines. Three peptides deduced from the 1.6-kb open reading frame reacted with human EBNA-positive sera, but not with EBNA-negative sera. Following affinity purification with the peptides, two of the corresponding human antibodies also reacted with the 87 000-dalton polypeptide.     

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.     

Antibodies directed against a synthetic peptide enable detection of a protein encoded by a vaccinia virus host range gene that is conserved within the Orthopoxvirus genus.

A vaccinia virus gene required for multiplication in some cell lines but not in others has been previously isolated and sequenced. A synthetic peptide predicted from the nucleotide sequence and corresponding to the carboxy-terminal 18 amino acids was used to raise antibodies in rabbits. The immune serum enabled detection of a 29-kilodalton (kDa) polypeptide by either immunoprecipitation or Western immunoblot assays. Synthesis of the 29-kDa polypeptide occurred immediately after infection and lasted for about 3 h. Shutoff of its synthesis was concomitant with the appearance of a delayed early polypeptide that may be antigenically related to the 29-kDa polypeptide. Analysis of cloned segments of the genomes of other orthopoxviruses by hybridization with the vaccinia virus host range gene demonstrates that it is well conserved within this genus.     

The DGA1 gene determines a second triglyceride synthetic pathway in yeast.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.     

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.     

Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria.

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.     

Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene.

The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.     

Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene.

The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.     

A synthetic intron in a naturally intronless yeast pre-tRNA is spliced efficiently in vivo.

Saccharomyces cerevisiae glutamine tRNA(CAG) is encoded by an intronless, single-copy gene, SUP60. We have imposed a requirement for splicing in the biosynthesis of this tRNA by inserting a synthetic intron in the SUP60 gene. Genetic analysis demonstrated that the interrupted gene produces a functional, mature tRNA product in vivo.     

Utilization of oxygen radicals by synthetic proline-rich oligopeptides]

Inhibition of superoxide by two synthetic proline-rich hexapeptides simulating the hinge region of the IgG molecule has been studied. The CPPPEL (P-Cys) peptide was active in utilizing superoxide (O2.-), while the APPPEL (P-Ala) peptide had no such activity. Spontaneous formation of clusters with six and/or eight monomers was shown for P-Cys, but not P-Ala. Preincubation of mixed P-Cys and P-Ala resulted in the appearance of a product with a high affinity for O2.-. Incubation of the mixture at 56 degrees C led to inactivation. Preincubation of P-Cys with ZnCl2 reversed the main pathway of O2.- utilization by P-Cys. The relationship of the P-Cys activity to its clusterization is discussed.